Expression of hepatitis B virus surface antigen P31 gene in Escherichia coli

A hepatitis B virus surface antigen (HBsAg) P31-coding DNA was constructed from a DNA fragment of the plasmid pHBr330 containing the entire hepatitis B virus (HBV) adr DNA and a chemically synthesized adaptor. The P31 gene was inserted into an expression vector, pTRP771, having an Escherichia coli tryptophan operon (trp) promoter to give a recombinant plasmid pTRP P31-R. The distance between the Shine-Dalgarno (SD) sequence and the initiation codon of P31 gene was adjusted to 9 bp. The expression level of HBsAg by E. coli 294[pTRP P31-R] was significantly elevated, in contrast to that of HBsAg by E. coli 294[pTRP SS-6]. Western blotting analysis has shown that E. coli[pTRP P31-R] synthesizes a specific polypeptide P31 of about 31 kDal, which reacts with anti-HBsAg antibody. The binding studies with polyalbumins from various species have also suggested that HBsAg P31 specifically binds to polymerized human serum albumin.     

Direct expression of hepatitis B surface antigen gene in E. coli

A 809 bp Sau 3A - Hpa I fragment containing a complete HBsAg gene and fragments 744 bp Hinc II - Hpa I and 712 bp Xba I - Hpa I containing a truncated HBsAg gene lacking the sequence encoding the NH2-terminal hydrophobic domain were prepared from a composite plasmid pHBV933 containing the 3.2 kb Eco RI DNA fragment of the entire HBV/adw genome and inserted into an expression vector pTRP801 to give plasmids pTRP SS-6, pTRP SS-39, and pTRP SS-50, respectively. The growth of a recombinant having pTRP SS-6 was greatly inhibited and the transformant expressed a low level of HBsAg, which is reactive to human anti-HBsAg antibody. Interestingly, the growth of transformants harbouring pTRP SS-39 and pTRP SS-50 was not inhibited and these transformants expressed a considerable level of the HBsAg. Minicells harbouring pTRP SS-6, pTRP SS-39, and pTRP SS-50 formed specific polypeptides of about 24 K, 23 K, and 22 K daltons, respectively.     

Efficient expression and characterization of hepatitis B virus preS2 antigen in Escherichia coli

The complete (encoding 55 amino acids, aa) or partial (encoding aa 1–26) preS2 region gene of hepatitis B virus (HBV) was fused to the 3-end of glutathion-S-transferase (GST) gene and expressed under the control of the inducible tac promoter in Escherichia coli at 37 °C. The fusion protein with the complete preS2 region was moderately expressed (8%) while the protein with the N-terminal 26 aa was expressed at a higher level, yielding about 20% of the total cellular proteins. The GST-preS2 (aa 1–26) protein, which contains the immunodominant epitope, was produced form the soluble protein fraction of the recombinant bacteria and purified by affinity chromatography using glutathione-agarose column. The purified preS2 fusion protein showed the antigenicity of preS2, as assessed by indirect and competitive ELISAs.     

Therapeutic inhibition of hepatitis B virus surface antigen expression by RNA interference

RNA interference (RNAi) mediated inhibition of virus-specific genes has emerged as a potential therapeutic strategy against virus induced diseases. Human hepatitis B virus (HBV) surface antigen (HBsAg) has proven to be a significant risk factor in HBV induced liver diseases, and an increasing number of mutations in HBsAg are known to enhance the difficulty in therapeutic interventions. The key challenge for achieving effective gene silencing in particular for the purpose of the therapeutics is primarily based on the effectiveness and specificity of the RNAi targeting sequence. To explore the therapeutic potential of RNAi on HBV induced diseases in particular resulted from aberrant or persistent expression of HBsAg, we have especially screened and identified the most potent and specific RNAi targeting sequence that directly mediated inhibition of the HBsAg expression. Using an effective DNA vector-based shRNA expression system, we have screened 10 RNAi targeting sequences (HBsAg-1 to 10) that were chosen from HBsAg coding region, in particular the major S region, and have identified four targeting sequences that could mediate sequence specific inhibition of the HBsAg expression. Among these four shRNAs, an extremely potent and highly sequence specific HBsAg-3 shRNA was found to inhibit HBsAg expression in mouse HBV model. The inhibition was not only preventive in cotransfection experiments, but also had therapeutic effect as assessed by post-treatment protocols. Moreover, this HBsAg-3 shRNA also exhibited a great potency of inhibition in transgenic mice that constitutively expressed HBsAg. These results indicate that HBsAg-3 shRNA can be considered as a powerful therapeutic agent on HBsAg induced diseases.     

Structure and expression of human hepatitis B virus surface antigen

In view of the growing occurrence rate of the virus-induced hepatitis B and also of the special role played by this particular virus (HBV) in the application of recombinant genetic techniques to the study of complex biological systems, an attempt was made to survey the available evidence concerning the widely investigated and practically the most important part of the viral genome, viz. the gene coding for the surface antigen (HBsAg) and the protein itself. The possible antigenic structure of the protein was investigated using data on the primary structure of 11 cloned HBsAg gene variants and on the synthesis of peptides simulating its immunological properties. Special emphasis was placed on quantitative assessment of antigenicity and immunogenicity. Expression of the gene in homologous systems was studied using cultures of eukaryotic tissues: both as part of HBV nucleotide sequences incorporated into the chromosome and as part of extrachromosomal DNA. The latest findings on HBsAg gene expression in yeast and bacteria are reviewed.     

Optimization of expression of hepatitis B surface antigen gene in yeasts

A series of recombinant plasmids has been constructed for expression of the hepatitis B viral surface antigen gene (HBsAg) under the control of the regulatory elements of the yeast acidic phosphatase gene (PHO5). The obtained plasmids possess the high mitotic and structural stability in the transformant yeast cells. The effect of different structural modifications of the vector on the level of HBsAg synthesis in yeasts has been studied. Optimal construction devoid of the bacterial DNA sequences and pre-S region has been selected.     

Expression of hepatitis B virus surface antigen P31 gene in yeast

The hepatitis B virus surface antigen (HBsAg) P31 gene has been expressed in yeast Saccharomyces cerevisiae. The gene products were shown to be glycoproteins with molecular sizes of 37,000 and 34,000 daltons (GP37 and GP34) containing polymerized albumin receptors. Successfully detecting these proteins depended on the extraction procedures. In the extract without protein denaturants and inhibitors, these products were degraded rapidly by proteases to yield smaller size derivatives lacking polymerized albumin receptors. As is the case in human serum-derived HBsAg, yeast HBsAg consisting of GP37 and GP34 was found to be particles or aggregates having a buoyant density of 1.2 g/cc; these particles bound to polymerized human serum albumin in species-specific manner.     

Distribution of insertion element IS1 in natural isolates of Escherichia coli

Total DNAs from twelve natural isolates of Escherichia coli from animals and humans were examined by hybridization with a probe for IS1. Considerable variation in copy number was found. In the case of two strains isolated from the same individual, one strain contained no copies of IS1 and the other, much greater than 30. Evidence was also obtained for the existence of IS1-like elements (iso-IS1s) of greater than 15% sequence divergence relative to the IS1 from antibiotic resistance plasmid R100.     

Transient expression of hepatitis B virus surface antigen (HBsAg) gene in monkey cells by a SV40-based virus vector

Summary We have constructed a recombinant SV40-based vector carrying the S gene coding for the hepatitis B virus surface antigen (HBsAg). This vector replicates as an episome in monkey COS7 cells, producing high levels of hepatitis B virus surface antigen (HBsAg), which is liberated in the cell medium, probably as a membrane vesicule. The vector also carries the SV40-late genes and produces recombinant viruses. These viruses were used to infect fresh cell culture, with detection of HBsAg in the medium. Thus, this virus vector can efficiently transduce the gene for HBsAg.     

Expression of hepatitis B virus surface antigen gene in Escherichia coli

Direct expression of hepatitis B virus (HBV) surface antigen (HBsAg) gene under the control of the Escherichia coli tryptophan operon (trp) promoter has been achieved. Synthesis of HBsAg (both complete and lacking its N-terminal segment) as a part of hybrid proteins with the N-terminal portion coded by genes cat, kan or bla is controlled by the appropriate promoters, as well as by the trp promoter. The highest levels of expression, including those for direct synthesis of HBsAg, provide the accumulation of about 10⁵ polypeptide molecules per bacterial cell.     

Efficient expression,purification and characterization of hepatitis B virus preS1 protein from Escherichia coli

Summary The complete (encoding 119 aminon acids, aa) or partial (encoding the N-terminal 90 aa) preS1 region gene of hepatitis B virus (HBV) was fused to the 3-end of glutathione-S-transferase (GST) gene and expressed at 37 °C under the control of the inducible tac promoter in E. coli. The results showed that the fusion protein with the full length of preS1 was moderately expressed, about 10% of total cellular proteins, while the protein with the partial preS1 was highly expressed, about 33% of total cellular proteins but the half was degraded into the protein with about N-terminal 60 aa of preS1. Accordingly, GST fusion protein containing the N-terminal 56 aa of the preS1, which still encodes B-and T-cell epitopes and a hepatocyte receptor binding site, was expressed under the same induction conditions and was shown to be highly and stably expressed, about 37% of total cellular proteins. The fusion protein with the full length or N-terminal 56 aa of preS1 and the peptides were simply and successfully purified by affinity chromatography and were demonstrated to exhibit the antigenicity and immunogenicity of the preS1 antigen.     

Synthesis in yeast of hepatitis B virus surface antigen modified P31 particles by gene modification

The pre-S2 portion of hepatitis B virus surface antigen P31 gene was modified to make gene products resistant to trypsin-like proteases in Saccharomyces cerevisiae. The coding sequence for 6 amino acids (Ser44 - Thr49) including Arg48 was removed, and the altered gene was inserted into an expression vector. The modified HBsAg P31 (M-P31c) gene products, consisting of GP37 and GP34, formed particles having both HBsAg antigenicity and polymerized-albumin receptor activity. Since the M-P31c particles can elicite two kinds of protective antibodies against hepatitis B virus, anti-S and anti-pre-S2 antibodies, the M-P31c particles are expected to be potentially effective to S-nonresponders.